Project Summary/Abstract: Sickle cell disease is accompanied by both chronic and severe episodic pain that is difficult to treat, and profoundly erodes the quality of life of those who suffer from it. Despite a detailed understanding of the genetics, molecular biology and biochemistry of sickle hemoglobin, the pathogenesis of the profound pain syndromes observed in sickle cell disease remain incompletely understood and likely involve complex and heterogeneous steps occurring in both the peripheral and central nervous systems. The goal of this proposal is to elucidate the mechanisms by which sickle cell disease results in pain, focusing on peripheral mechanisms in primary afferent nerve terminals. Using a murine model of severe sickle cell disease, the Berkeley Sickle Mice, we demonstrate that these mice exhibit marked hypersensitivity to mechanical, heat and cold peripheral stimuli. Furthermore, induction of acute sickling with hypoxia specifically exacerbates the ongoing mechanical hypersensitivity in sickle cell mice. In agreement, teased fiber recordings from skin-nerve preparations from these mice indicate that both myelinated A fiber and unmyelinated C fiber nociceptors are sensitized to mechanical stimuli. These findings parallel the mechanical hypersensitivity and pain reported by patients with sickle cell disease. Thus, these sickle cell mice represent a novel model of long-lasting chronic pain hypersensitivity that is closely associated with a human disease. On the basis of these findings and our observations with sensory plasticity in other pain models, we hypothesize that sensitization of primary afferent terminals contributes to sickle cell pain and that this sensitization is mediated by increased function of Transient Receptor Potential ion channels. Therefore, the Specific Aims for this project are to 1) Characterize the sensitization state of primary afferent fibers to mechanical, heat and cold stimuli in mice with sickle cell disease. 2) Determine the contribution of the Transient Receptor Potential (TRP) Ion Channels TRPA1 and TRPV1 to both the behavioral hypersensitivity and the sensitization of primary afferent fibers in sickle cell disease. 3) Characterize how acute vaso-occlusion modulates mechanical hypersensitivity in sickle mice. We will use both ex vivo and in vivo electrophysiological recordings to characterize the sensitization state of primary afferent fibers in Berkeley sickle cell mice. Next, we will utilize both genetic (TRP channel null mice induced with sickle cell disease) and pharmacologic approaches (selective TRP channel antagonists) to determine the role of specific TRP-family ion channels in sickle cell-associated primary afferent sensitization and pain behavior. Finally, we will induce acute sickling crises by an experimental model of vaso-occlusion to study how vaso-occlusion modulates mechanical hypersensitivity in sickle mice. These interrelated Specific Aims provide a multifaceted, coordinated and tightly focused approach that will clarify the role of primary afferent neurons in the development of pain syndromes within the complex setting of sickle cell-induced vascular and organ pathologies, as well as provide insight into the potential value of targeted TRP antagonist therapies for sickle cell pain.